Annals of Agricultural and Environmental Medicine 2021, Vol 28, No 3, 397–403 ORIGINAL ARTICLE www.aaem.pl Analysis of selected serological parameters in patients with diagnosed Lyme borreliosis and in seropositive patients with no clinical symptoms Małgorzata Tokarska-Rodak1,A,C-F , Anna Pańczuk1,C-E , Hanna Fota-Markowska2,3,A-B,F , Katarzyna Matuska4,B-C 1 Faculty of Health Sciences, Pope John Paul II State School of Higher Education, Biala Podlaska, Poland 2 Institute of Rural Health, Lublin, Poland 3 Department of Infectious Diseases, Skubiszewski Medical University, Lublin, Poland 4 Department of Microbiological Diagnostics Clinical Hospital No. 1, Lublin, Poland A – Research concept and design, B – Collection and/or assembly of data, C – Data analysis and interpretation, D – Writing the article, E – Critical revision of the article, F – Final approval of article Tokarska-Rodak M, Pańczuk A, Fota-Markowska H, Matuska K. Analysis of selected serological parameters in patients with diagnosed Lyme borreliosis and in seropositive patients with no clinical symptoms. Ann Agric Environ Med. 2021; 28(3): 397–403. doi: 10.26444/aaem/124088 Abstract Objectives. The aim of the study was to analyze some metalloproteinases, cytokines, and chemokines in LB patients and healthy seropositive subjects. The presence of IgM/IgG antibodies against specific Borreliella antigens was analyzed in the presence or absence of clinical manifestations of LB. Materials and method. The study involved 38 patients diagnosed with LB and arthralgia and/or arthritis symptoms, and 57 foresters presenting no clinical symptoms of LB. The ELISA test was applied for general screening of anti-Borreliella IgM/IgG. Western blot was used for confirmatory diagnosis of LB for the positive and borderline results. Serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF, IL-8, CCL5, CXCL9/MIG, CCL2/MCP-1, CXCL10/IP-10 concentrations were measured with the use of the Human Cytometric CBA test. The concentration of MMP-2 and MMP-9 in the serum was determined with the use of ELISA tests. Results. Analysis of the cytokines and chemokines revealed that only the concentration of IL-2 was significantly higher (2.4 pg/m; p=0.00641) in patients with LB symptoms than in the seropositive individuals (0.4 pg/ml). The MMP2 concentration was significantly higher (233.3 ng/ml; p=0.00294) in patients with clinical manifestations of LB than in those occupationally exposed to tick bites, but did not have anti-Borreliella antibodies (192.0 ng/ml). Conclusions. The presence of IgG antibodies against a number of Borreliella antigens and the differences in the IL-2 and MMP2 levels in seropositive or seronegative individuals and symptomatic LB patients, may indicate differences in the intensity of the immune response to the infection and, consequently, may induce development of clinical manifestations of the disease in seropositive and seronegative individuals. Key words cytokines, MMP-9, MMP-2, Lyme borreliosis, Borreliella INTRODUCTION B. burgdorferi seem to be the most neurotropic and the most arthritogenic species, respectively [3]. Lyme borreliosis (LB) is caused by spirochetes of the Lyme In accordance with current recommendations, the Disease (LD) group of the Borreliaceae species which are presence of at least one of the clinical symptoms: erythema transmitted by ticks [1]. LB is a disease with diverse clinical migrans, borrelial lymphoma, and acrodermatitis chronica presentations the most common of which is the manifestation athrophicans, or a set of symptoms comprising Lyme of erythema migrans (EM). However, the infecting pathogen carditis, Lyme arthritis, and neuroborreliosis, is the basis for can spread to other tissues and organs, causing more severe the diagnosis of Lyme borreliosis. Another indispensable manifestations involving the patient’s skin, nervous system, element of diagnostics (besides EM skin lesions localized joints, or heart [2, 3, 4]. early) is the detection of specific anti-Borreliella antibodies. More than 10 genospecies are included to LD group of Laboratory diagnostics consists of two stages: detection Borreliaceae species: Borreliella afzelii, Borreliella garinii, of specific antibodies with the enzyme immunoassay and Borreliella bavariensis, Borreliella burgdorferi, and occasionally confirmation with the Western blot test [6, 7, 8]. Borreliella spielmanii and Borreliella lusitaniae, are pathogenic An interesting phenomenon is the presence of specific anti- to humans in Europe, whereas Borreliella burgdorferi and, Borreliella antibodies without clinical signs of infection [9–19]. in certain areas, Borreliella mayonii, are human pathogens Detection of antibodies alone does not evidence the disease in North America [1, 5]. The spirochetes B. afzelii is mostly [5, 7]. The reasons why some patients present clinical signs of associated with skin manifestations and B. garinii and Lyme borreliosis while others only undergo seroconversion are not fully known. It seems that the type of generated immune Address for correspondence: Anna Pańczuk, Faculty of Health Sciences, Pope John Paul II State School of Higher Education, Biala Podlaska, Poland reactions may have key importance for the development of E-mail: [email protected] Lyme borreliosis. Neutrophils, acidophils, mast cells, and Received: 25.03.2020; accepted: 17.06.2020; first published: 08.07.2020 macrophages are involved in the immune response against 398 Annals of Agricultural and Environmental Medicine 2021, Vol 28, No 3 Małgorzata Tokarska-Rodak, Anna Pańczuk, Hanna Fota-Markowska, Katarzyna Matuska. Analysis of selected serological parameters in patients with diagnosed… of Borreliella species. The inflammatory response mediators The reaction wells were coated with a mixture of antigens produced by these cells, as well as the synergistic interactions derived from B. burgdorferi, B. afzelii, and B. garinii, as well of macrophages and T cells, influence the intensity of immune as the recombinant B. burgdorferi VlsE antigen. Results below response to infection [20]. B. burgdorferi spirochetes do 16 relative units/ml (RU/ml), between 16 – 22 RU/ml, and not secrete enzymes degrading the extracellular matrix above 22 RU/ml were regarded as negative, borderline, and that would facilitate entrance into the host organism and positive, respectively. migration in tissues [21]. However, they are able to activate The Western blot Anti-Borrelia EUROLINE-WB IgM test proteolytic enzymes, e.g. matrix metallproteinases (MMPs), (Euroimmun, Germany) was used for confirmation of the and thus penetrate human tissues [22]. Metalloproteinases positive and borderline IgM results. The test strips contained a are involved in degradation of protein components of the complete B. afzelii antigen extract and a membrane chip with extracellular matrix and hydrolysis of molecules released from recombinant VlsE antigen. In the IgG class, an Anti-Borrelia the cell surface. Thus, they can activate or inactivate many EUROLINE-RN-AT-IgG kit (Euroimmun, Germany) was cytokines, chemokines, and growth factors [23, 24]. Cytokines employed as a confirmation test. The test strips contained secreted by activated T cells have a considerable impact on the highly specific recombinantB. burgdorferi s.s. antigens (p83, regulation and effectiveness of immune response. Th1 cells p58, p21, p20, p19, p18), highly specific recombinant dimeric secrete IFN-g and TNF, thus promoting cytotoxic phagocyte- OspC (advance, p25) from B. burgdorferi, B. garinii, and dependent immune response. In turn, Th2 cells secrete IL-4, B. afzelii, purified recombinant flagellin (p41) and BmpA IL-5, and IL-9 inducing humoral phagocyte-independent (p39) from B. afzelii, Borrelia afzelii lipid (LBa), B. burgdorferi. immune response [10]. lipid, and highly purified recombinant VlsE antigens. Serological parameters: cytokines and metalloproteinases. OBJECTIVES Serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-g, and TNF concentrations were measured with the use of the Human The aim of the study is to analyze some serological parameters Cytometric Bead Array Kit Th1/Th2/Th17 test (Becton (metalloproteinases, cytokines, and chemokines) in Lyme Dickinson; BD). The level of IL-8 (CXCL8), CCL5 (RANTES, borreliosis patients and healthy seropositive subjects. The Regulated on Activation, Normal T-cell Expressed and presence of IgM/IgG antibodies against specific Borreliella Secreted), monokine induced by interferon gamma (CXCL9/ antigens was analyzed in the presence or absence of clinical MIG), monocyte chemoattractant protein-1 (CCL2/MCP- manifestations of Lyme borreliosis. 1), and interferon gamma-induced protein-10 (CXCL10/ IP-10) in serum samples were determined using the Human Chemokine I Cytometric Bead Array Kit (Becton Dickinson; MATERIALS AND METHODS BD). Equipment: FACSCantoTMII cytometer and FCAP ArrayTM Software Version 3.0. (Becton Dickinson; BD). Study group. The examinations involved 95 subjects, The concentration of MMP-2 in the serum was determined including: 38 patients diagnosed with Lyme borreliosis and (ELISA, R&D): a monoclonal antibody specific for MMP-2 arthralgia and/or arthritis symptoms, who were patients in has been pre-coated onto a microplate. Standards and samples the Clinic for Infectious Diseases at the Medical University, in were pipetted into the wells and any MMP-2 present was Lublin, eastern Poland. The diagnosis was based on medical bound by the immobilized antibody. Minimum detectable history, physical examination, clinical picture, and serological